A consensus molecular subtypes classification strategy for clinical colorectal cancer tissues.

Consensus Molecular Subtype (CMS) classification of colorectal cancer (CRC) tissues is complicated by RNA degradation upon formalin-fixed paraffin-embedded (FFPE) preservation. Here, we present an FFPE-curated CMS classifier. The CMSFFPE classifier was developed using genes with a high transcript integrity in FFPE-derived RNA. We evaluated the classification accuracy in two FFPE-RNA datasets with matched fresh-frozen (FF) RNA data, and an FF-derived RNA set. An FFPE-RNA application cohort of metastatic CRC patients was established, partly treated with anti-EGFR therapy. Key characteristics per CMS were assessed. Cross-referenced with matched benchmark FF CMS calls, the CMSFFPE classifier strongly improved classification accuracy in two FFPE datasets compared with the original CMSClassifier (63.6% versus 40.9% and 83.3% versus 66.7%, respectively). We recovered CMS-specific recurrence-free survival patterns (CMS4 versus CMS2: hazard ratio 1.75, 95% CI 1.24-2.46). Key molecular and clinical associations of the CMSs were confirmed. In particular, we demonstrated the predictive value of CMS2 and CMS3 for anti-EGFR therapy response (CMS2&3: odds ratio 5.48, 95% CI 1.10-27.27). The CMSFFPE classifier is an optimized FFPE-curated research tool for CMS classification of clinical CRC samples.

Total neoadjuvant therapy with or without aflibercept in rectal cancer: 3-year results of GEMCAD-1402.

BACKGROUND: The results of the Grupo Español Multidisciplinar en Cáncer Digestivo (GEMCAD)-1402 phase II randomized trial suggested that adding aflibercept to modified fluorouracil, oxaliplatin, and leucovorin (mFOLFOX6) induction, followed by chemoradiation and surgery, could increase the pathological complete response (pCR) rate in patients with high-risk, locally advanced rectal cancer. Here we update results up to 3 years of follow-up and evaluate the predictive value of consensus molecular subtypes identified with immunohistochemistry (IHC). METHODS: Patients with magnetic resonance imaging-defined T3c-d and/or T4 and/or N2 rectal adenocarcinoma in the middle or distal third were randomly assigned to mFOLFOX6 induction, with aflibercept (mF+A; n = 115) or without aflibercept (mF; n = 65), followed by capecitabine plus radiotherapy and surgery. The risk local relapse, distant metastases, disease-free survival (DFS), and overall survival (OS) were estimated at 3 years. Selected samples were classified via IHC into immune-infiltrate, epithelial, or mesenchymal subtypes. RESULTS: mF+A and mF had 3-year DFS of 75.2% (95% confidence interval [CI] = 66.1% to 82.2%) and 81.5% (95% CI = 69.8% to 89.1%), respectively; 3-year OS of 89.3% (95% CI = 82.0% to 93.8%) and 90.7% (95% CI = 80.6% to 95.7%), respectively; 3-year cumulative local relapse incidences of 5.2% (95% CI = 1.9% to 11.0%) and 6.1% (95% CI = 1.7% to 15.0%), respectively; and 3-year cumulative distant metastases rates of 17.3% (95% CI = 10.9% to 25.5%) and 16.9% (95% CI = 8.7% to 28.2%), respectively. pCRs were achieved in 27.5% (n = 22 of 80) and 0% (n = 0 of 10) of patients with epithelial and mesenchymal subtypes, respectively. CONCLUSION: Adding aflibercept to mFOLFOX6 induction was not associated with improved DFS or OS. Our findings suggested that consensus molecular subtypes identified with IHC subtypes could be predictive of pCR with this treatment.

Long-term Survival Update and Extended RAS Mutational Analysis of the CAIRO2 Trial: Addition of Cetuximab to CAPOX/Bevacizumab in Metastatic Colorectal Cancer.

BACKGROUND: Here we present updated survival of the CAIRO2 trial and assessed whether the addition of anti-EGFR to anti-VEGF therapy could still be an effective treatment option for patients with extended RAS/BRAF wildtype and left-sided metastatic colorectal cancer (mCRC). MATERIALS AND METHODS: Retrospective updated survival and extended RAS and BRAF V600E mutational analysis were performed in the CAIRO2 trial, a multicenter, randomized phase III trial on the effect of adding cetuximab to a combination of capecitabine, oxaliplatin (CAPOX), and bevacizumab in mCRC. RESULTS: Updated survival analysis confirmed that the addition of cetuximab did not provide a benefit on either progression free (PFS) or overall survival (OS) in the intention-to-treat population. With the extended mutational analyses 31 KRAS, 31 NRAS and 12 BRAF V600E additional mutations were found. No benefit of the addition of cetuximab was observed within the extended wildtype group, even when selecting only left-sided tumors (PFS HR 0.96, p = 0.7775). However, compared to the original trial an increase of 6.5 months was seen for patients with both extended wildtype and left-sided tumors (median OS 28.6 months). CONCLUSION: Adding cetuximab to CAPOX and bevacizumab does not provide clinical benefit in patients with mCRC, even in the extended wildtype group with left-sided tumors. However, in the extended wildtype group we did observe clinically relevant higher survival compared to the initial trial report, indicating that it is important to analyze a broader panel of RAS and BRAF variants using more recent sequencing techniques when assessing survival benefit after anti-EGFR therapy.

Molecular characterization of colorectal cancer related peritoneal metastatic disease.

A significant proportion of colorectal cancer (CRC) patients develop peritoneal metastases (PM) in the course of their disease. PMs are associated with a poor quality of life, significant morbidity and dismal disease outcome. To improve care for this patient group, a better understanding of the molecular characteristics of CRC-PM is required. Here we present a comprehensive molecular characterization of a cohort of 52 patients. This reveals that CRC-PM represent a distinct CRC molecular subtype, CMS4, but can be further divided in three separate categories, each presenting with unique features. We uncover that the CMS4-associated structural protein Moesin plays a key role in peritoneal dissemination. Finally, we define specific evolutionary features of CRC-PM which indicate that polyclonal metastatic seeding underlies these lesions. Together our results suggest that CRC-PM should be perceived as a distinct disease entity.

Serum-based measurements of stromal activation through ADAM12 associate with poor prognosis in colorectal cancer.

BACKGROUND: Recently it has been recognized that stromal markers could be used as a clinically relevant biomarker for therapy response and prognosis. Here, we report on a serum marker for stromal activation, A Disintegrin and Metalloprotease 12 (ADAM12) in colorectal cancer (CRC). METHODS: Using gene expression databases we investigated ADAM12 expression in CRC and delineated the source of ADAM12 expression. The clinical value of ADAM12 was retrospectively assessed in the CAIRO2 trial in metastatic CRC with 235 patients (31% of total cohort), and an independent rectal cancer cohort (n = 20). RESULTS: ADAM12 is expressed by activated CRC associated fibroblasts. In the CAIRO2 trial cohort, ADAM12 serum levels were prognostic (ADAM12 low versus ADAM12 high; median OS 25.3 vs. 17.1 months, HR 1.48 [95% CI 1.11-1.96], P = 0.007). The prognostic potential was specifically high for metastatic rectal cancer (HR 1.78 [95% CI 1.06-3.00], P = 0.030) and mesenchymal subtype tumors (HR 2.12 [95% CI 1.25-3.60], P = 0.004). ADAM12 also showed potential for predicting recurrence in an exploratory analysis of non-metastatic rectal cancers. CONCLUSIONS: Here we describe a non-invasive marker for activated stroma in CRC which associates with poor outcome, especially for primary cancers located in the rectum.

Development of a miRNA-based classifier for detection of colorectal cancer molecular subtypes.

Previously, colorectal cancer (CRC) has been classified into four distinct molecular subtypes based on transcriptome data. These consensus molecular subtypes (CMSs) have implications for our understanding of tumor heterogeneity and the prognosis of patients. So far, this classification has been based on the use of messenger RNAs (mRNAs), although microRNAs (miRNAs) have also been shown to play a role in tumor heterogeneity and biological differences between CMSs. In contrast to mRNAs, miRNAs have a smaller size and increased stability, facilitating their detection. Therefore, we built a miRNA-based CMS classifier by converting the existing mRNA-based CMS classification using machine learning (training dataset of n = 271). The performance of this miRNA-assigned CMS classifier (CMS-miRaCl) was evaluated in several datasets, achieving an overall accuracy of ~ 0.72 (0.6329-0.7987) in the largest dataset (n = 158). To gain insight into the biological relevance of CMS-miRaCl, we evaluated the most important features in the classifier. We found that miRNAs previously reported to be relevant in microsatellite-instable CRCs or Wnt signaling were important features for CMS-miRaCl. Following further studies to validate its robustness, this miRNA-based alternative might simplify the implementation of CMS classification in clinical workflows.

Clinical Value of Consensus Molecular Subtypes in Colorectal Cancer: A Systematic Review and Meta-Analysis.

BACKGROUND: The consensus molecular subtypes (CMSs) of colorectal cancer (CRC) capture tumor heterogeneity at the gene-expression level. Currently, a restricted number of molecular features are used to guide treatment for CRC. We summarize the evidence on the clinical value of the CMSs. METHODS: We systematically identified studies in Medline and Embase that evaluated the prognostic and predictive value of CMSs in CRC patients. A random-effect meta-analysis was performed on prognostic data. Predictive data were summarized. RESULTS: In local disease, CMS4 tumors were associated with worse overall survival (OS) compared with CMS1 (hazard ratio [HR] = 3.28, 95% confidence interval = 1.27 to 8.47) and CMS2 cancers (HR = 2.60, 95% confidence interval = 1.93 to 3.50). In metastatic disease, CMS1 consistently had worse survival than CMS2-4 (OS HR range = 0.33-0.55; progression-free survival HR range = 0.53-0.89). Adjuvant chemotherapy in stage II and III CRC was most beneficial for OS in CMS2 and CMS3 (HR range = 0.16-0.45) and not effective in CMS4 tumors. In metastatic CMS4 cancers, an irinotecan-based regimen improved outcome compared with oxaliplatin (HR range = 0.31-0.72). The addition of bevacizumab seemed beneficial in CMS1, and anti-epidermal growth factor receptor therapy improved outcome for KRAS wild-type CMS2 patients. CONCLUSIONS: The CMS classification holds clear potential for clinical use in predicting both prognosis and response to systemic therapy, which seems to be independent of the classifier used. Prospective studies are warranted to support implementation of the CMS taxonomy in clinical practice.

The recurring features of molecular subtypes in distinct gastrointestinal malignancies-A systematic review.

In colorectal cancer (CRC), pancreatic ductal adenocarcinoma (PDAC) and gastric cancer (GC) multiple studies of inter-tumor heterogeneity have identified molecular subtypes, which correlate with clinical features. Our aim was to investigate the attributes of molecular subtypes across three different gastrointestinal cancer types. We performed a systematic search for publications on molecular subtypes or classifications in PDAC and GC and compared the described subtypes with the established consensus molecular subtypes of CRC. Examining the characteristics of subtypes across CRC, PDAC and GC resulted in four categories of subtypes. We describe uniting and distinguishing features within a mesenchymal, an epithelial, an immunogenic and a metabolic and digestive subtype category. We conclude that molecular subtypes of CRC, PDAC and GC display relevant overlap in molecular features and clinical outcomes. This finding encourages quantitative studies on subtypes across different cancer types and could lead to a paradigm shift in future treatment strategies.

Molecular subtype-specific efficacy of anti-EGFR therapy in colorectal cancer is dependent on the chemotherapy backbone.

BACKGROUND: Patient selection for addition of anti-EGFR therapy to chemotherapy for patients with RAS and BRAF wildtype metastatic colorectal cancer can still be optimised. Here we investigate the effect of anti-EGFR therapy on survival in different consensus molecular subtypes (CMSs) and stratified by primary tumour location. METHODS: Retrospective analyses, using the immunohistochemistry-based CMS classifier, were performed in the COIN (first-line oxaliplatin backbone with or without cetuximab) and PICCOLO trial (second-line irinotecan with or without panitumumab). Tumour tissue was available for 323 patients (20%) and 349 (41%), respectively. RESULTS: When using an irinotecan backbone, anti-EGFR therapy is effective in both CMS2/3 and CMS4 in left-sided primary tumours (progression-free survival (PFS): HR 0.44, 95% CI 0.26-0.75, P = 0.003 and HR 0.12, 95% CI 0.04-0.36, P < 0.001, respectively) and in CMS4 right-sided tumours (PFS HR 0.17, 95% CI 0.04-0.71, P = 0.02). Efficacy using an oxaliplatin backbone was restricted to left-sided CMS2/3 tumours (HR 0.57, 95% CI 0.36-0.96, P = 0.034). CONCLUSIONS: The subtype-specific efficacy of anti-EGFR therapy is dependent on the chemotherapy backbone. This may provide the possibility of subtype-specific treatment strategies for a more optimal use of anti-EGFR therapy.

Apc-mutant cells act as supercompetitors in intestinal tumour initiation.

A delicate equilibrium of WNT agonists and antagonists in the intestinal stem cell (ISC) niche is critical to maintaining the ISC compartment, as it accommodates the rapid renewal of the gut lining. Disruption of this balance by mutations in the tumour suppressor gene APC, which are found in approximately 80% of all human colon cancers, leads to unrestrained activation of the WNT pathway1,2. It has previously been established that Apc-mutant cells have a competitive advantage over wild-type ISCs3. Consequently, Apc-mutant ISCs frequently outcompete all wild-type stem cells within a crypt, thereby reaching clonal fixation in the tissue and initiating cancer formation. However, whether the increased relative fitness of Apc-mutant ISCs involves only cell-intrinsic features or whether Apc mutants are actively involved in the elimination of their wild-type neighbours remains unresolved. Here we show that Apc-mutant ISCs function as bona fide supercompetitors by secreting WNT antagonists, thereby inducing differentiation of neighbouring wild-type ISCs. Lithium chloride prevented the expansion of Apc-mutant clones and the formation of adenomas by rendering wild-type ISCs insensitive to WNT antagonists through downstream activation of WNT by inhibition of GSK3ß. Our work suggests that boosting the fitness of healthy cells to limit the expansion of pre-malignant clones may be a powerful strategy to limit the formation of cancers in high-risk individuals.

Tumour budding is associated with the mesenchymal colon cancer subtype and RAS/RAF mutations: a study of 1320 colorectal cancers with Consensus Molecular Subgroup (CMS) data.

BACKGROUND: Tumour budding is an important prognostic factor in colorectal cancer (CRC). Molecular profiling of tumour buds suggests (partial) epithelial-mesenchymal transition and cancer stem-cell phenotype, similarly described in the "mesenchymal" Consensus Molecular Subtype 4 (CMS4), which identifies a particularly poor prognostic subgroup. Here, we determine the association of tumour budding with CMS classification, prognosis, and response to therapy. METHODS: AMC-AJCCII-90 cohort (n = 76, stage II) was evaluated for peritumoural budding on H&E slides. LUMC (n = 270, stage I-IV), CAIRO (n = 504, metastatic CRC) and CAIRO2 (n = 472, metastatic CRC) cohorts were investigated for intratumoural budding using pan-cytokeratin-stained tissue microarrays. Budding was scored as count/area, then classified as <5 or ≥5 buds. For all cohorts, CMS classifications were available (gene-expression/immunohistochemistry-based classifiers). RESULTS: High (≥5) budding predicted a worse outcome in multivariate analysis in AMC-AJCCII-90 (p = 0.018), LUMC (p < 0.0001), and CAIRO (p = 0.03), and in CAIRO2 (continuous variable, p = 0.02). Tumour budding counts were higher in CMS4 compared to epithelial CMS2/3 cancers (p < 0.01, all), and associated with KRAS/BRAF mutations (p < 0.01, AMC-AJCCII-90, CAIRO, CAIRO2). CONCLUSION: Tumour budding is an adverse prognostic factor across all CRC stages and is associated with the mesenchymal CMS4 phenotype. KRAS/BRAF mutations are strongly correlated with tumour budding suggesting their involvement in the regulation of this process.

Classification of Colorectal Cancer in Molecular Subtypes by Immunohistochemistry.

Colorectal cancer (CRC) is a heterogeneous disease, which can be categorized into distinct consensus molecular subtypes (CMSs). These subtypes differ in both clinical as well as biological properties. The gold-standard classification strategy relies on genome-wide expression data, which hampers widespread implementation. Here we describe an immunohistochemical (IHC) Mini Classifier, a practical tool that, in combination with microsatellite instability testing, delivers objective and accurate scoring to classify CRC patients into the main molecular disease subtypes. It is a robust immunohistochemical-based assay containing four specific stainings (FRMD6, ZEB1, HTR2B, and CDX2) in combination with cytokeratin. We also describe an online tool for classification of individual samples based on scoring parameters of these stainings.